Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β1b or β2a subunits, but not with the β3 or β4 subunits (Grueter et al. 2008). CaMKII-dependent facilitation of CaV1.2 LTCCs requires Thr498 phosphorylation in the β2a subunit (Grueter et al. 2006), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain CaV1.2α1 and β1 or β2 subunits, but is not detected in LTCC complexes containing β4 subunits. CaMKIIα can be specifically tethered to the I/II linker of CaV1.2 α1 subunits in vitro by the β1b or β2a subunits. Efficient targeting of CaMKIIα to the full-length CaV1.2α1 subunit in transfected HEK293 cells requires CaMKII binding to the β2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β2a at Thr498 within the LTCC complex, without altering overall phosphorylation of CaV1.2α1 and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.

The midbrain dopamine system located within the ventral tegmental area (VTA) and substantia nigra (SN) contain dopaminergic (DA) neurons that are known to mediate various goal directed behaviors. Although research with animals provides a framework for understanding DA function, the differential role of midbrain VTA and SNc in normal human brain function remains to be elucidated. Functional Magnetic Resonance Imaging (fMRI) at lower field strengths (1.5 T and 3 T) has demonstrated blood oxygen level dependent (BOLD) responses in the human midbrain. However, these studies lacked the sensitivity and resolution to distinguish activation within the VTA and SN. FMRI at ultra-high fields (7 T and higher) has been shown to provide better spatial resolution and higher sensitivity for BOLD signal contrast, suggesting its suitability for assessing VTA and SN functions in humans. The aims of this thesis were to delineate the structural architecture and the functional significance of the human midbrain DA system using 7 T MRI. High resolution anatomical images revealed fine details of structures within the midbrain, emphasizing the iron rich SN and red nuclei, as well as microvasculature. These images enabled detailed volumetric analysis of the midbrain nuclei in healthy humans. A reward-related task was used in the functional studies that probed both reward anticipation and reward outcome behavioral constructs. However, we failed to observe significant BOLD-related activity in the midbrain across participants for selected different MR sequences in the functional studies. The low significance of task-related signal changes was examined in detail and it was concluded that the MR signal variance in the midbrain was problematically high. Various algorithms designed to mitigate known noise sources did not significantly reduce this variance. Preliminary evidence that some signal variations in the midbrain may reflect physiological baseline fluctuations of neural origin deserves further investigation. Overall, this thesis has demonstrated the value of ultra-high field MRI for evaluating structural changes associated with neurodegenerative diseases pertinent to the midbrain, and has comprehensively evaluated the challenges of functional studies of this important brain region.

Attention-Deficit Hyperactivity Disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain imaging studies suggest a contribution of altered dopamine (DA) signaling to ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at pre- and post-synaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified human DAT (hDAT) coding variants, A559V located in the transmembrane domain 12, a region thought to play a role in DAT oligomerization, and R615C, located in the transporters distal C-terminus, a region previously implicated in constitutive and regulated transporter trafficking. The A559V variant exhibits an anomalous DA efflux sustained by the actions of cell surface dopamine D2 receptor and hyperphosphorylation as well as altered AMPH-mediated trafficking. Detailed characterization of the R615C (DAT 615C) variant demonstrates that whereas wildtype DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively. Moreover, DAT 615C exhibits insensitivity to the endocytic effects of AMPH and protein kinase C (PKC) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered calcium/calmodulin-dependent protein kinase II (CaMKII) and flotillin-1 interactions. Using C-terminal peptides derived from wildtype DAT and the R615C variant, we provide evidence that the DAT 615C C-terminus can act dominantly to preclude AMPH regulation of wildtype DAT. Mutagenesis of DAT C-terminal sequences suggest that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies reinforce the utility of rare gene variant studies in neuropsychiatric disorders, they support a coupling of DAT microdomain localization with transporter regulation and they provide evidence of perturbed DAT-mediated DA signaling as a risk determinant for ADHD.

The thalamic caudal intralaminar nuclear complex is composed of the centromedian-parafascicular (CM-PF) nuclei in primates and the PF in rodents. These thalamic neurons project to the striatum and synapse onto the dendritic shafts and spines of medium spiny neurons (MSNs). In Parkinsons disease (PD), nigrostriatal dopamine neurons and CM-PF neurons degenerate. We evaluated if substantia nigra (SN) dopamine neurons project to the PF and determined if loss of nigrostriatal dopamine neurons causes the degeneration of PF neurons in rats. We found that the SN projection to the PF is non-dopaminergic. A previous study reported that 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, which contains dopaminergic and noradrenergic axons, decreased the number of retrogradely-labeled PF thalamostriatal neurons. Given the lack of an SN dopaminergic innervation of the PF, we hypothesized that the loss of retrograde labeling was secondary to forebrain noradrenergic depletion. However, we observed no significant differences in retrograde labeling or in the total number of PF neurons in rats with nigrostriatal dopamine or forebrain noradrenergic depletion. Thus, it appears that the loss of CM-PF neurons is not caused by the transsynaptic degeneration of catecholaminergic neurons in PD. We next sought to determine how lesions of the PF alter striatal MSNs. However, conventional lesions of the PF invariably damage surrounding nuclei. We searched for genes encoding proteins that were selectively expressed in the PF. We found that Cbln1 was enriched in the PF of rats. Virtually all PF neurons expressed Cbln1, and ultrastructural studies revealed that Cbln1-immunoreactive axon terminals formed axodendritic and axospinous synapses with MSNs. Previous studies indicated that Cbln1 regulates the formation and maintenance of cerebellar synapses. We therefore hypothesized that a loss of Cbln1 in thalamostriatal neurons would modify the dystrophic changes in MSN dendritic spines seen in the dopamine-depleted (parkinsonian) striatum. We found that in the cbln1 null mutant mouse, MSN spine density was markedly increased, with a corresponding increase in the density of axospinous synapses. Our findings suggest that modulation of Cbln1 may be a novel means of slowing progression in PD.

For two principle reasons, the transmembrane Met receptor tyrosine kinase has emerged as an important molecular target for study in the developing forebrain: 1) it regulates a variety of neurodevelopmental processes in vitro, including cell migration, neurite outgrowth, and synaptogenesis, and 2) replicate human genetic studies have demonstrated associations of allelic MET variants with autism spectrum disorders (ASD). One particular MET variant reduces transcriptional efficiency in vitro, and MET protein levels are reduced ~2-fold in postmortem ASD neocortex, implicating reduced MET signaling in the etiology of this neurodevelopmental disorder. To begin to clarify the neurodevelopmental influences of MET signaling in vivo, we comprehensively mapped Met mRNA and Met protein expression in the mouse forebrain throughout perinatal and postnatal development. In situ hybridization revealed Met transcript expression throughout the cerebral cortex and in limbic structures including the hippocampus, amygdala, and septum. Met immunohistochemistry showed Met protein enrichment in long-projecting axons of neurons within these forebrain structures during peak periods of axon arborization and synaptogenesis over the first two postnatal weeks. Comparative immunohistochemical mapping in the nonhuman primate macaque demonstrated conserved temporal and subcellular patterns of Met expression. Spatially, Met protein expression was conserved in subcortical limbic structures, but highly restricted neocortically within the cingulate gyrus and temporal lobes. Collectively, these data implicate Met signaling in the wiring of cortical and limbic circuits, which govern species-typical social and emotional behaviors that are atypical in ASD. Moreover, they predict circuit-level, presynaptic consequences of Met signaling disruption. Single-cell morphometric analyses in a forebrain-specific conditional Met knockout mouse confirmed this, revealing discrete effects on dendrite and dendritic spine morphology. In summary, these studies reveal a role for Met signaling in the development of forebrain connectivity and further our understanding of selective circuit vulnerabilities in ASD. Future efforts will employ electrophysioligical and biochemical/bioinformatics approaches to elucidate the functional consequences of developmental Met signaling disruptions in the forebrain.

The epithalamus of the zebrafish contains the dorsal habenulae (Dh), a bilateral pair of nuclei characterized by significant left-right asymmetries in gene expression, anatomy, and connectivity. A screen for mutations that affected habenular laterality identified a nonsense mutation in the sec61a-like 1(sec61al1) gene, which codes for the alpha subunit of the ER translocon, a vital entry point for the maturation and processing of all transmembrane and many secreted proteins. sec61al1 mutants undergo an altered program of neurogenesis, producing excess early-borne neurons of the lateral subnucleus at the expense of the later-borne medial subnucleus. Ultrastructural analysis of the cells lining the 3rd ventricle indicates that periventricular precursor cells, which form an epithelium in wild-type embryos, lose apical-basal polarity in sec61al1 mutants. Furthermore, trafficking of N-cadherin through the Sec61 translocon likely mediates differentiation of dorsal habenula progenitor cells, as morphants depleted for cdh2 (the gene that encodes the N-cadherin protein), also develop symmetric LsDh and cdh2 mutant progenitor cells preferentially contribute to the LsDh class. We conclude that Sec61al1 acts to maintain structural polarity in habenular precursor cells, in turn regulating the timing of asymmetric neurogenesis in the habenular nuclei: in the absence of Sec61al1, progenitor cells of the lateral subnucleus overproliferate and differentiate, ultimately resulting in symmetric habenular nuclei.

The spinal Neuropeptide Y (NPY) system is a potential target for development of new pain therapeutics. NPY and two of its receptors (Y1 and Y2) are found in the superficial dorsal horn of the spinal cord, a key area of nociceptive gating and modulation. Lumbar intrathecal injection of Neuropeptide Y (NPY) is antinociceptive, reducing hyper-reflexia to thermal and mechanical stimulation, particularly after nerve injury and inflammation. We have previously shown that intrathecal injection of the targeted cytotoxin, Neuropeptide Y-sap (NPY-sap), is also antinociceptive, reducing nocifensive reflex responses to noxious heat and formalin. In the present study, we sought to determine the role of dorsal horn Y1R-expressing neurons in pain by destroying them with NPY-sap and testing the rats on three operant tasks. We also sought to determine the extent and selectivity of the lesion by staining tissue from rats injected with other peptide-saporin conjugates, Derm-sap and Gal-sap, for the Y1 receptor, and, conversely, staining the NPY-sap tissue for MOR and Gal-R1. Lumbar intrathecal NPY-sap 1- reduced CFA-induced hyper-reflexia on the 10°C cold plate, 2- reduced cold aversion on the thermal preference and escape tasks, 3- was analgesic to noxious heat on the escape task, 4- reduced the CFA-induced allodynia to cold temperatures experienced on the thermal preference, feeding interference, and escape tasks, 5- did not inhibit or interfere with morphine analgesia, and 6- reduced immunoperoxidase staining for Y1R in the superficial dorsal horn. These data indicate that Y1R-expressing dorsal horn neurons play an important role in pain modulation, particularly after peripheral inflammation. The involvement of Y1R-expressing neurons in modulating cold pain and the observation that intrathecal injection of NPY-sap does not interfere with morphine analgesia, along with our previous findings that intrathecal NPY-sap doesnt affect protective reflexes, pose Y1R-expressing dorsal horn neurons as excellent candidates to be targeted for the development of analgesic drugs.

Audition is critical for communication and survival, yet the cortical pathways and mechanisms by which complex sounds are processed remain poorly understood. From the results of a number of studies, a working model of primate auditory cortex has emerged, where it is proposed to contain three regions: core, belt, and parabelt. These three regions can be thought of as levels of processing, and are subdivided into multiple areas, distinguished by unique anatomical and physiological profiles. Anatomical connections between regions and areas have suggested a hierarchy of processing, but the direction and flow of information within and between regions and areas remains an active area of study. Here, we characterize the response properties of neurons in a number of areas across all three regions of the auditory cortex of awake macaques. We find that response latencies are consistent with the flow of information in two directions: across regional level (core-belt-parabelt) and across areas in a caudal to rostral direction. Evidence from response tuning to temporally modulated frequencies also is consistent with a direction of flow across regional level. Though there is evidence of directional flow in multiple areas, it is clear that areas are responding within a very close timescale to each other. Lastly, an analysis of pairwise correlations within and across areas suggests auditory cortex is organized into a weakly yet isotopically connected functional network, where effective connectivity is stronger within an area than across areas. This series of studies is the most complete coverage of multiple areas in primate auditory cortex in the literature to date. Converging evidence from both physiology and anatomy points to an emerging understanding that primate auditory cortex processes sound in multiple areas with a strong degree of parallel processing. This is an architecture optimally designed for the processing of rapid time-based stimuli such as sound.

The proper function of the nervous system is dependent upon a delicate balance between excitatory and inhibitory activity in the brain. GABAA receptors are extremely important in the maintenance of this balance because they mediate the majority of fast inhibition in the adult central nervous system. Several genetic mutations in various human GABAA receptor subunits have been associated with idiopathic generalized epilepsy syndromes. Here we have investigated the consequences of GABAA receptor dysfunction using a mouse model based on one of the aforementioned genetic mutations identified in a human epilepsy patient. We begin with a general introduction to epilepsy followed by a more detailed discussion of the particular epilepsy syndrome studied herein. We then provide a thorough review of GABAA receptor structure and function and highlight previous findings related to our current studies. We then provide an explanation of the rationale and general experimental strategy employed in our studies. Next we provide the specifics of our methodology proceeded by the presentation of our results. In the final chapter we discuss our interpretation of the data and its implications for the advancement of our understanding of epilepsy.

A myriad of human behaviors, such as mood, awareness and motivation, are modulated by the monoamine neurotransmitters serotonin, norepinephrine and dopamine, respectively. Consequently, dysfunction of these monoaminergic systems underlies numerous medical conditions. In particular, disturbances in the serotonergic system are implicated in depression, bipolar disorder, and autism, whereas the dopaminergic system is implicated in Parkinsonâs disease and addiction. During neurotransmission high concentrations of monoamine neurotransmitters are released from presynaptic neurons into the synaptic cleft where they diffuse to bind and activate pre- and postsynaptic receptors. The primary way to terminate neurotransmission involves monoamine transporters, which shuttle monoamines back into presynaptic neurons where they replenish synaptic vesicle contents. The monoamine transporters are molecular targets for antidepressants and psychostimulants that function to increase monoamine levels in the brain. For example, serotonin transporter (SERT) reuptake is inhibited by Prozac to increase serotonin levels and treat various mood disorders. Similarly, dopamine transporter (DAT) reuptake is altered with drugs, such as cocaine or amphetamine, which results in enhanced dopaminergic signaling and is thought to underlie reward and addictive behaviors. Transport through the monoamine transporters is not thoroughly understood, and the traditional model with fixed substrate-ion stoichiometry has been challenged in recent years with the discovery of ionic currents mediated by monoamine transporters. In an effort to better understand the activity of monoamine transporters, a variety of substrates and inhibitors are utilized. In particular, in my work I characterize fluorescent compounds that are based on a known monoamine transporter substrate and describe their utility as reporters to study serotonin transporter activity in real-time. In addition, I describe a novel effect induced by amphetamine and related compounds at both DAT and SERT whereby even after external removal of these compounds, a persistent current remains. These studies provide information about various substrates that exert an array of distinct effects on SERT and DAT, which may enable further studies to elucidate the nature of transporter biophysics. 1. APP+ is a Fluorescent Substrate for the Serotonin Transporter One limitation to transporter research is the inability to monitor substrate uptake in real-time. Traditional methods such as radiolabeled uptake assays, though highly specific, yield poor temporal resolution. Electrophysiology on the other hand provides excellent time resolution, but currents are mediated mostly by ionic fluxes and therefore do not yield direct information about substrate transport. To investigate this issue, we collaborated with Dr. Ian D. Tomlinson and the laboratory of Dr. Sandra Rosenthal to develop compounds based on a known monoamine transporter substrate. We identified and characterized a fluorescent compound called APP+ that is suitable to monitor SERT transport in real-time. We employed a range of techniques to elucidate thoroughly the specificity of this compound for SERT expressed in Xenopus laevis oocytes and mammalian cells. Finally, we used APP+ to study binding and transport through SERT. This work will help to uncover fundamental information about hSERT, and to improve our ability to study these transporters. 2. Amphetamine Induces a Persistent Leak Current in the Dopamine Transporter Amphetamine (AMPH) and related compounds increase dopamine (DA) levels in the brain and cause profound behavioral effects. One target for these drugs is the dopamine transporter (DAT) that normally regulates synaptic DA levels. DAT agonists, such as DA and AMPH, induce DAT-mediated currents driven by sodium. By measuring DAT currents on voltage-clamped Xenopus laevis oocytes, we discovered a DAT leak current induced by external exposure to the S(+)amphetamine (S(+)AMPH) enantiomer that persists long after its removal. We determined that the AMPH-induced leak current in DAT depends on sodium and is blocked by cocaine. In addition, intracellular application of S(+)AMPH can induce the leak current effectively, which suggests an internal secondary binding site in DAT. Understanding this novel effect of amphetamine on DAT has implications in the understanding of human behavior because amphetamine-induced persistent currents likely impact dopaminergic signaling, DA release mechanisms, and amphetamine abuse. 3. A Comparison of Leak and Persistent Leak Currents Induced by Methamphetamine and 3,4-Methylenedioxymethamphetamine in the Human Dopamine and Serotonin Transporters After establishing the S(+)amphetamine-induced persistent leak current at DAT, we expanded this work to test if other DAT-mediated releaser agents related to AMPH would also induce the persistent leak current. In particular, we focused on 3,4-methylenedioxy-methamphetamine (MDMA) and methamphetamine (METH) because it is known that although MDMA and METH are structurally related they exert distinct behavioral effects in people. However, since MDMA has preference for the serotonin transporter and METH acts more potently at the dopamine transporter, we made a comparison of the effects of METH and MDMA on both SERT and DAT. Lastly, we uncovered that the amphetamine derivative para-chloroamphetamine (pCA) confers a substantial persistent leak current at the human serotonin transporter. These findings could open new avenues towards the study of the effect drugs of abuse have on behavior.